ABSTRACT: Calcific aortic valve disease (CAVD) is the most common cause of aortic stenosis, affecting 25% of people over the age of 65 years. Patients who progress to symptomatic, severe aortic valve stenosis have a 50% mortality within 2 years if left untreated by surgical or transcatheter aortic valve replacement (TAVR). Currently, there is no medical therapy for the prevention or treatment of this CAVD. Moreover, surgery can be a high-risk procedure for many older patients with multiple comorbidities. Inflammatory signaling appears to play a key role in progression of calcification and may hold the potential to develop the first therapeutic treatment of CAVD. However, the underlying mechanisms of inflammatory calcification are minimally understood. Our laboratory has begun defining a novel inflammatory signaling pathway critical to progressive vascular calcification in models of hypercholesterolemia. We determined macrophage Rac1-dependent expression of the inflammatory cytokine, interleukin-1? (IL-1?), is required for progressive atherosclerotic calcification. The role of macrophage Rac1-mediated IL-1? expression has never been evaluated in models of calcific aortic valve disease. Unpublished preliminary data presented in this proposal demonstrates that this Rac1-induced IL-1? expression can be upregulated in macrophages under conditions consistent with hypercholesterolemia-induced CAVD. Using a hyperlipidemia-based mouse model (ApoE-/-) for CAVD and an inducible macrophage Rac1-deletion (CSF1RmcmRac1fl/fl), we observed in a preliminary study that the development of CAVD was dependent on macrophage-specific Rac1 signaling. Echocardiographic measures for aortic valve stenosis, including elevated blood flow velocity and valve area, were decreased in mice after inducible gene-deletion of macrophage Rac1. Histological assessment of the valve demonstrated reduced valve thickness, decreased valve calcification and decreased fibrosis in the macrophage Rac1-deleted animals. Moreover, we observed reduced circulating (serum) IL-1? levels in the macrophage Rac1-deleted animals. Our group's previous work has indicated that IL-1? promotes bone-forming osteogenic transcription factor expression and is strongly associated with vascular calcification in a macrophage Rac-dependent manner. Considering observations of macrophage Rac1 on CAVD development, it's association with IL-1?, and past findings on the contribution of IL-1? to calcification in vitro, we hypothesize that a macrophage- specific Rac1-IL-1? signaling pathway is a critical inflammatory mechanism that drives the progression of CAVD to severe aortic valve stenosis. Our specific aims are to determine the individual roles of both Rac1 activity and IL-1? in the promotion of CAVD and consequent aortic valve stenosis in vivo. Defining mechanisms of CAVD is critical to the development of novel therapeutic interventions aimed at disrupting the natural course of the disease.